Developing inhibitory peptides against SARS-CoV-2 envelope protein

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has affected approximately 800 million people since the start of the Coronavirus Disease 2019 (COVID-19) pandemic. Because of the high rate of mutagenesis in SARS-CoV-2, it is difficult to develop a sustainable approach for prevention and treatment. The Envelope (E) protein is highly conserved among human coronaviruses. Previous studies reported that SARS-CoV-1 E deficiency reduced viral propagation, suggesting that E inhibition might be an effective therapeutic strategy for SARS-CoV-2. Here, we report inhibitory peptides against SARS-CoV-2 E protein named iPep-SARS2-E. Leveraging E-induced alterations in proton homeostasis and NFAT/AP-1 pathway in mammalian cells, we developed screening platforms to design and optimize the peptides that bind and inhibit E protein. Using Vero-E6 cells, human-induced pluripotent stem cell-derived branching lung organoid and mouse models with SARS-CoV-2, we found that iPep-SARS2-E significantly inhibits virus egress and reduces viral cytotoxicity and propagation in vitro and in vivo. Furthermore, the peptide can be customizable for E protein of other human coronaviruses such as Middle East Respiratory Syndrome Coronavirus (MERS-CoV). The results indicate that E protein can be a potential therapeutic target for human coronaviruses.

The authors show data both in vitro and in vivo that look intriguing.Overall, the paper shows that the N-terminal domain of the E protein in SARS-2 CoV reverses changes in lysosome fluorescence and host/viral protein expression caused by E overexpression or viral infection.I find the results showing the changes caused by the peptide convincing, although the mechanism by which these are happening are not entirely clear.
We appreciate the reviewer's positive comments on the beneficial effect of iPep-SARS2-E.
It appears that the DD mutant has a direct interaction with E protein, but how is this linked to the events observed is more challenging to explain.The readouts used are sequence dependent: a double D mutant has a better effect than the WT, whereas another mutant chosen shows almost no effect.
The DD mutant was found to have higher activity, and is used in subsequent experiments.If the hypothesis of interaction is correct, this mutant should have higher affinity for E than the WT sequence of the peptide, and even more for the negative control used.Can the authors show some experimental data that this is the case?By SPR/BLI for example?The authors use electrophysiological recordings, but I am unable to comment if the current observed is due to E. The experiment may be far more convincing with a negative control E mutant which has no channel activity (N15A: refs PMID: 24788150, PMID: 22832120).
We thank this reviewer for these comments on the molecular mechanism underlying the effect of iPep-SARS2-E (TAT-MY18-2ED).Regarding the electrophysiological recording, we have shared our plasmid DNA with various electrophysiologists, and we confirmed the reproducibility in other laboratories using mammalian cell lines (unpublished) and oocyte expression system (Harrison et al. Commun. Biol. 2022, doi: 10.1038/s42003-022-03669-2.).In a separate manuscript, we plan to report the effect of a variety of mutants on the electrophysiological properties of Envelope proteins.
To address the hypothesis that the DD mutant (E7D & E8D, we called 2ED mutant) may have higher affinity for SARS-CoV-2 E (2E) protein than the wild-type, we conducted computational modeling of the full-length 2E pentameric structure with iPep-SARS2-E using AlphaFold2 ColabFold model (DeepMind).However, plDDT value of the provided structure and interaction was so low (<50, not shown) that we could not conclude and finalize their interaction interface and difference between the wild-type and 2ED mutant peptides.For setting up binding assays, next we expressed and tried to purify bacterial recombinant 2E protein:

1) His-Envelope and Envelope-His protein constructs:
We used pCold vector (TaKaRa Bio/Clontech) to express His-tagged full-length 2E proteins in Rosetta 2(DE3) bacteria strain (two versions, N-and C-terminal His-tagged).Unfortunately, the protein expression was low in bacteria and the yield was low, requiring silver gel staining, and the majority of 2E proteins were found in the flowthrough fraction, suggesting that His-tag addition to the ends might not be suitable to purify the full-length 2E proteins.Also, we found that the N-and C-terminus of His-2E proteins might be degraded/ truncated (Fig. R1a,b, previous page).

2) Thrombin cleavable construct:
Following the results using 2E-His and His-2E constructs, next we used the same pCold vector, thrombin cleavage site and super-folder CFP (sfCFP)-His to improve the expression, binding to Ni column and yield and to purify the full-length 2E protein using Ni column and thrombin.sfCFP was thought to be helpful when fluorescent chromatography is used, according to our previous experiences.The 2E fusion protein expression became higher in bacteria, and the yield was improved, allowing us to observe the protein bands using CBB staining (Fig. R2a).However, the majority of 2E fusion proteins were still found in the flowthrough fractions, and 2E protein truncation/degradation happened.Though we also examined the effect of further proteinase inhibitor addition on the purification steps, such 2E protein truncation/degradation still happened (not shown).Although thrombin treatment produced 2E protein, the efficiency and yield were not satisfying to obtain the full-length 2E protein (Fig. R2b).While we have successfully expressed and purified other recombinant proteins in bacteria (Bekdash et al. Cell Rep. Methods 2021, PMID: 35475001), for this manuscript revision, unfortunately, we could not prepare the purified recombinant full-length 2E protein sample suitable for binding assays.
Alternatively, we conducted co-immunoprecipitation using His-tagged MY18 wild-type and 2ED mutant peptides to pull down the full-length 2E proteins using mammalian heterologous expression system, although it'd be not as quantitative as SPR/BLI.The results demonstrate there was no obvious difference in 2E protein pull-down efficiency between the wild-type and 2ED mutant peptides (Fig. R3), though we tested two different con-transfection ratios (2E-YFP: His-MY18, 1:1 and 1:3, not shown), while ELISA demonstrates that there was significant difference in the affinity to anti 2E-N N2A5E8 mAb between the wildtype and 2ED mutant peptides (Fig. S2c).
We really appreciate this reviewer's advice, which enabled us for taking various approaches to address the question.We truly thank this reviewer for providing the helpful comment and advice.

Fig. R1 |
Fig. R1 | Recombinant Envelope proteins using bacterial expression system and Hig-tag/Ni column purification.Representative sliver stain and immunoblot images of SARS-CoV-2 Envelope (2E) protein using His tag: 2E-His (a) and His-2E constructs (b).Though 2E protein is composed of 75 amino acids, the band was localized between 10kDa and 15kDa protein ladders; SDS sample buffer contains 4M urea.

Fig. R2 |
Fig. R2 | Applying thrombin cleavage site for His tagmediated 2E protein purification.(a) Representative CBB stain and immunoblot images of 2E proteins with thrombin cleavage site (TC), sfCFP and His tag.(b) Thrombin treatment to cleave the fusion protein.Representative CBB stain and immunoblot images of the treated 2E proteins are shown.